Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protein synthesis because it was unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H+-ATPase inhibitor. The fate of internalized 1251-SP was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface.

Understanding the physiological role of tachykinins requires precise cellular and subcellular localization of their receptors. We raised antisera by immunizing rabbits with peptides corresponding to portions of the intracellular tails of the rat neurokinin 1, 2, and 3 receptors (NK1-R, NK2-R, NK3-R). Receptors were localized by immunofluorescence and confocal microscopy. NK1-R, NK2-R, and NK3-R were detected at the plasma membrane of transfected cells with minimal intracellular stores. Staining was abolished by preabsorption of the antisera with the peptides used for immunization. Nontransfected cells were unstained. Each antiserum only stained cells transfected with the appropriate receptor and did not stain cells transfected with the other receptors. Therefore, the antisera are specific and do not cross-react with other neurokinin receptors. We examined the distribution of the neurokinin receptors in the gastrointestinal tract of the rat. NK1-R was detected in myenteric and submucosal neurons and in interstitial cells of Cajal. NK2-R was localized to circular and longitudinal muscle cells and to nerve endings in the plexuses. NK3-R was detected in numerous myenteric and submucosal neurons. Some neurons expressed both NK1-R and NK3-R. Receptors were detected at the plasma membrane and in endosomes. Cells expressing the receptors were closely associated with tachykinin-containing nerve fibers. Thus, NK1-R and NK3-R mediate neurotransmission by tachykinins within enteric nerve plexuses, and NK1-R and NK2-R mediate the effects of tachykinins on interstitial and smooth muscle cells, respectively.

Proteases regulate cells by cleaving proteinase‐activated receptors (PARs). Thrombin and trypsin cleave PAR‐1 and PAR‐2 on neurons and astrocytes of the brain to regulate morphology, growth and survival. We hypothesized that thrombin and mast cell tryptase, which are generated and released during trauma and inflammation, regulate enteric neurons by cleaving PAR‐1 and PAR‐2. We detected immunoreactive PAR‐1 and PAR‐2 in > 60 % of neurons from the myenteric plexus of guinea‐pig small intestine in primary culture. A large proportion of neurons that expressed substance P, vasoactive intestinal peptide or nitric oxide synthase also expressed PAR‐1 and PAR‐2. We confirmed expression of PAR‐1 and PAR‐2 in the myenteric plexus by RT‐PCR using primers based on sequences of cloned guinea‐pig receptors. Thrombin, trypsin, tryptase, a filtrate from degranulated mast cells, and peptides corresponding to the tethered ligand domains of PAR‐1 and PAR‐2 increased [Ca2+]i in > 50 % of cultured myenteric neurons. Approximately 60 % of neurons that responded to PAR‐1 agonists responded to PAR‐2 agonists, and > 90 % of PAR‐1 and PAR‐2 responsive neurons responded to ATP. These results indicate that a large proportion of myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors also express PAR‐1 and PAR‐2. Thrombin and tryptase may excite myenteric neurons during trauma and inflammation when prothrombin is activated and mast cells degranulate. This novel action of serine proteases probably contributes to abnormal neurotransmission and motility in the inflamed intestine.

In the digestive system, substance P is an excitatory transmitter to muscle, a putative excitatory neuro-neuronal transmitter, a vasodilator, and a mediator in inflammatory processes. Many of the biological effects of substance P are mediated by a high-affinity interaction with the tachykinin receptor neurokinin-1. The aim of the present study was to identify the sites of expression of this receptor in the rat stomach and intestine by immunohistochemistry with a polyclonal antiserum raised to the intracellular C-terminal portion of the rat neurokinin-1 receptor. Neurokinin-1 receptor immunoreactivity is present in a large population of enteric neurons. The relative density of these neurons along the gut is colon > ileum >> stomach. In the intestine, stained neurons have a smooth cell body with processes that can be followed within and between plexuses, and make close approaches to other neuronal cells, but do not appear to project outside the plexuses, suggesting that they are interneurons. In the stomach, neurokinin-1 receptor-immunoreactive neurons are infrequent and have a poorly defined and irregular shape. Neurokinin-1 receptor immunoreactivity is also localized to numerous non-neuronal cells in the inner portion of the circular muscle layer of the small intestine, which have the appearance of small dark smooth muscle cells or interstitial cells of Cajal. These cells are postulated to form a "stretch-sensitive" system with the deep muscular plexus and thus constitute an important site of regulation of muscle activity. Double labeling immunofluorescence was used to simultaneously localize neurokinin-1 receptor and substance P/tachykinin immunoreactivities. These experiments demonstrate that in the enteric plexuses, substance P/tachykinin-immunoreactive varicose fibers encircle the cell bodies of most neurokinin-1 receptor-containing neurons, and in the inner portion of the circular muscle layer of the small intestine they lie close to neurokinin-1 receptor-immunoreactive non-neuronal cells. In addition, some enteric neurons express both neurokinin-1 receptor and substance P/tachykinin immunoreactivities. The present study provides strong evidence that the neurokinin-1 receptor is the tachykinin receptor mediating the actions of substance P on enteric neurons and smooth muscle.

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